اثر خاموشی ژن چالکون ایزومراز بر مسیر تولید رنگ در گیاه اطلسی با استفاده از فناوری RNAi

نوع مقاله : مقالات پژوهشی

نویسندگان

• فاطمه کیخا آخر ¹
• عبدالرضا باقری ²
• نسرین مشتاقی ³
• مسعود فخرفشانی ⁴

¹ گروه ژنتیک و تولید گیاهی، دانشکده کشاورزی، دانشگاه جهرم، جهرم، ایران

² گزوه بیوتکنولوژی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران

³ گروه بیوتکنولوژی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران

⁴ گروه ژنتیک و تولید گیاهی، دانشگاه جهرم، جهرم، ایران

https://doi.org/10.22067/jhs.2022.76190.1162

چکیده

رنگ گل یکی از مهمترین خصوصیات گیاهان زینتی است که نقش ویژه ای را در فروش این نوع گیاهان ایفا می­کند. در سال­های اخیر با استفاده از فناوری­های نوین از قبیل مهندسی ژنتیک ارقام جدیدی وارد بازار شده است که یکی از موثرترین روش‌های آن، کاهش مقدار رنگدانه‌های داخلی گل بوسیله ممانعت از فعالیت آنزیم‌های ضروری مورد نیاز برای بیوسنتز آن‌ها می‌باشد. روش RNAi یک فناوری نوین خاموشی ژن است که امکان بررسی ژن‌های دخیل در تولید رنگ گل را فراهم کرده است. در این پژوهش، با استفاده از این فناوری نوین، نقش ژن چالکون ایزومراز (chi) به عنوان یکی از ژن‌های کلیدی در مسیر بیوسنتزی آنتوسیانین‌ها مورد بررسی قرار گرفت. در این آزمایش سازه RNAi ژن chi طراحی شد و به اطلسی­های با رنگ گل بنفش و صورتی انتقال یافت. لاین‌های تراریخته، تغییرات متنوعی را در رنگ گل نشان دادند و به گروه­های فنوتیپی متفاوتی تقسیم شدند. این تغییرات بیشتر به صورت کاهش مقدار تجمع رنگدانه‌ها و کمرنگ شدن گل‌ها بود. با بررسی بیان ژن chi، کاهش معنی‌دار بیان این ژن نیز مشاهده شد. بطور کلی نتایج این تحقیق نشان داد که خاموشی ژن با استفاده از فناوری RNAi می‌تواند روش مناسبی برای کاهش بیان ژن باشد به طوری که با استفاده از این روش می­توان به نقش ژن­های مختلف در مسیرهای بیوسنتزی ترکیبات از جمله آنتوسیانین­ها پی برد. علاوه بر این، ژن چالکون ایزومراز به عنوان یکی از ژن‌های موثر در مسیر بیوسنتزی آنتوسیانین‌ها در گیاه اطلسی شناخته شد که می‌تواند در تولید رنگ در این گیاه نقش داشته باشد.

کلیدواژه‌ها

• آنتوسیانین
• اطلسی
• چالکون ایزومراز
• خاموشی ژن
• RNAi

20.1001.1.20084730.1402.37.2.9.5

موضوعات

• بیوتکنولوژی و اصلاح درختان و گل های زینتی

عنوان مقاله [English]

The Effect of Chalcone Isomerase Gene Silencing on Pigment Production Pathway in Petunia hybrida with RNAi Technology

نویسندگان [English]

• Fatemeh Keykha Akhar ¹
• Abdolreza Bagheri ²
• Nasrin Moshtaghi ³
• Masud Fakhrfeshani ⁴

¹ Department of Plant Genetic and Production, Faculty of Agriculture, Jahrom University, Jahrom, Iran

² Department of Biotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

³ Department of Biotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

⁴ Department of Plant Genetic and Production, Faculty of Agriculture, Jahrom University, Jahrom, Iran

چکیده [English]

Introduction
 Flower color is one of the most significant characteristics in ornamental plant breeding. New varieties of various plants in relation to their flower color have been obtained by monitoring the expression levels of genes involved or regulating the flavonoid and anthocyanin biosynthesis pathway. Flavonoids possess significant and diverse biological functions. They are the major pigments for flowers, fruits, seeds, and leaves. They are natural products that contain a C6-C3-C6 carbon framework and are synthesized by a branched pathway that yields both colored and colorless compounds. The gene encoding chalcone isomerase (CHI) is among the genes and enzymes identified in the flavonoid pathway. This enzyme catalyzes the isomerization of naringenin chalcone into the corresponding flavanone. CHI enzyme belongs to the family of isomerases, specifically the class of intramolecular lyases. Chalcone isomerase has a core 2-layer alpha/beta structure and has attracted much attention recently due to its role in stress response and pigment production. One of the most effective methods of genetic engineering is the reduction of flower pigments by suppression of required enzymes for their biosynthesis. RNA interference (RNAi) has provided the tool for the investigation of genes involved in the production of flower color. Silencing of any gene in the anthocyanin biosynthetic pathway can result in reduced or inhibited anthocyanin production. RNAi technology is an effective gene silencing method and a powerful tool for studying gene function and development of new traits by transformation of viral RNA or hairpin RNA (hpRNA) constructs into plants. The processing of dsRNA into 21-23-nt small interfering RNAs (siRNAs), and the mediators of RNAi, triggers cognate mRNA degradation. The hpRNAi methodology simply requires a transgene construct containing an inversely-repeated sequence of the target gene flanked with a promoter and terminator which effectively function in plants.
 
Material and Methods
 In this research, with the design and construction of chiRNAi, the transformation of the RNAi construct was carried out of Petunia plants. Potted plants of P. hybrida were grown under standard greenhouse conditions (16-17°C night temperature and 21-24°C day temperature and photoperiod 16/8 (light/dark)). The RNAi construct including the 530 bp cds of the chalcone isomerase (chi) gene and 741 bp of pdk gene as intron between chi sense and antisense were used for transient RNAi-induced silencing. The pBI121-chi530 plasmids were introduced into A. tumefaciens strain LBA4404 by electroporation method. Colonies of A. tumefaciens carrying the desired plasmid were screened by PCR with specific primers for chi gene. RNAi construct co-cultured with petunia’s leave. Samples was kept in dark condition for 3 days and then transferred to branch induction media. Samples were investigated for phenotypical changes and chi gene expression by qRT-PCR.
Results and Discussion
 Transgenic lines showed a reduced number of pigments and a faded flower color. So that, in purple petunia, was shown 5 phenotypical groups. These groups was indicated different levels of chi gene silencing. In pink petunia was seen two groups of phenotypical changes. In these plants, chi-RNAi construct was reduced pigment production and so, these plants had faded colors in petals. Also, the chi gene expression was reduced in all transgenic lines. Generally, the results of this research showed that RNAi can be used as an efficient method for gene silencing. The application of gene silencing can indicate the gene’s function in biosynthesis pathways of various components such as anthocyanins. In addition, the chalcone isomerase gene was identified as one of the effective genes in anthocyanin biosynthesis pathway in Petunia plants that could be involved in the production of color in these plants; hence, chi gene silencing resulted in clear phenotypic alterations in this plant.
 
Conclusion
 In general the concentration of the target mRNA in a particular tissue could be a factor that influences silencing efficiency. At very low levels of gene expression, small amounts of the silencing target, mRNA, could be completely degraded by the RNA-induced silencing complex (RISC), whereas the presence of higher amounts of the target mRNA may result in incomplete silencing, allowing some residual functional mRNA to be translated into the corresponding protein. This research demonstrated the hpRNA construct has been successfully established for floral tissues of P. hybrida. The hpRNA construct was developed for chi-RNAi silencing of one of the key genes in the anthocyanin biosynthetic pathway in Petunia flowers. The silencing of the chi gene is a prototype for the modification of the anthocyanin biosynthetic pathway in Petunia through gene suppression. This strategy could also be useful for rapid functional analysis of other genes involved in flower development.
 

کلیدواژه‌ها [English]

• Anthocyanin
• Chalcone isomerase
• Gene silencing
• Petunia hybrida
• RNAi

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